Implantation of the early embryo requires the interaction between the maturing embryonic trophectoderm and maternal endometrium. This requires exquisite control of differentiation and communication between the two tissues. The overall goal of pilot project II is to dissect the role of microRNAs in the post-transcriptionnal regulation of trophoblast differentiation. This pilot is a two year collaborative project based on the expertise of the members and cores of the proposed center for trophoblast differentiation, implantation, and early development. To address the overall goal of the project, a genetic model that specifically knocks-out microRNA function has been developed. This model will be used to phenotypically characterize the role of miRNAs in trophoblast determination, differentiation, and function. The specific aims of the grant include: 1) characterization of the role of microRNAs in pre-implantation development. This will include their role in earliest cell fate choice defining the trophectoderm versus the epiblast as well the role of microRNAs in the producing a healthy blastocyst that is able to attach and interface with the matenal endometrium. 2) characterization of the role of microRNAs in later stages of trophectoderm development including the maintenance and differentiation of trophoblast stem cells as well as the capacity of knockout trophectoderm to invade and migrate through the endometrial matrix. This work will use a conditional microRNA knockout model that the lab has developed enabling us to bypass earlier defects in development caused by premature loss of miRNAs. This aim will also determine the differential expression of microRNAs throughout trophectoderm development. This pilot project will act as a springboard to future experiments and R01-type grants aimed at understanding specific developmental pathways regulated by miRNAs, which then be used diagnostically and therapeutically to follow and treat patients with defects in embryonic implantation. MicroRNAs are a newly discovered class of cell regulators whose pervasiveness and global effects on gene function suggests they have a central role in both normal and abnormal human development. This has already been borne out for diseases such as cancer and likely will be the case in reproductive disease as well. Completion of this pilot grant will provide initial insights into the role of microRNAs in normal reproductive development necessary to dissect their role as well as identify potential diagnostic and therapeutic targets in reproductive disease.